Zhihui Deng

HLA-C polymorphisms and PCR dropout in exons 2 and 3 of the Cw*0706 allele in sequence-based typing for unrelated Chinese marrow donors

To evaluate the accuracy of SBT protocols for HLA-C and to better understand the HLA-C polymorphism in Chinese, 1795 unrelated CMDP donors were typed at exons 2, 3, and 4 of the HLA-C gene using the Atria commercial kit. Of the study subjects, 1768 showed conclusive typing results, whereas the other 27 showed inconclusive results. Subsequent full-length cloning and haplotype sequencing showed that 11 of the 27 inconclusive results could be explained by the presence of nine novel alleles identified: Cw*0130, 0624, 070206, 075602, 0766, 0767, 0820, 0821, and 0827. These novel alleles were generated by a total of 10 coding-region substitutions, eight of them being located in the antigen-binding groove. Cw*0766 and Cw*075602 were detected three and two times, respectively, in the 1795 donors. The other 16 inconclusive samples were retested by SBT using our in-house PCR primers; all of them were found to carry Cw*0706, which dropped out in exons 2 and 3 in the initial PCR using the commercial primers amplifying from 5 UTR to intron 3. Our results showed the importance of the full-length genomic sequence and intronic SNPs for the development of more accurate SBT. The allele distribution and novel alleles detected in this study also provide further insights into the HLA-C polymorphism in the Chinese Han population.

Genetic profile of KIR and HLA in southern Chinese Han population

KIR and their HLA ligands are encoded by two of the most diverse gene families in the human genome. The function of KIR on the NK cell is highly dependent on the normal expression of class I HLA on the target cell. Previous population studies in southern Chinese have been focused on the KIR framework genes and genotypes but little is known about the compound profiles of KIR/HLA. The present study examined 503 unrelated individuals from southern Chinese Han population for the polymorphism of KIR and class I HLA genes. All 16 KIR genes were detected in the study population and the four framework genes KIR3DL2, 3DL3, 3DP1, and 2DL4 were present in all individuals. Thirty unique KIR gene profiles were found reflecting a rather limited number of KIR haplotypes in this population. KIRAA1 was the most common profile observed in 54.7% of the samples. Among the AA1 individuals, 15.6% were homozygous for the deleted KIR2DS4. Haplotype A (74.8%) was more common than haplotype B (25.2%). HLA-C1 was a much more common ligand for 2D KIRs than C2. Bw4-80I, Bw4-80T, and the Bw4-bearing HLA-A alleles were detected at similar frequencies. The matched KIR+HLA pairs 2DL2/3+C1 (98.1%), 3DL1+Bw4 (73.3%), 3DL2+A3/11 (60.0%) were the most common ones whereas 3DS1+Bw4-80I was the least common (9.4%). A total of 193 unique compound profiles of KIR-HLA were identified in 480 informative individuals, 130 of the profiles being detected only once. The study provided a comprehensive analysis of the KIR/HLA profiles in southern Chinese in regards of the presence/absence of KIR genes, HLA ligands, matched KIR+HLA pairs, and KIR/HLA compound profiles. The results could help to better understand the role played by KIR/HLA interaction in associated diseases and clinical transplantation in southern Chinese.

Congenital Tetragametic Blood Chimerism Explains a Case of Questionable Paternity

Abstract:u2002 Human chimerism is the presence of ≥2 cell populations in one person that contain genetic material from more than one zygote. Chimerism may be either acquired by transfusion or transplantation of donor cells, or congenital arising from embryo fusion or dizygotic twin–twin transfusion. We encountered a 4‐year‐old boy with developmental hip dysplasia whose preoperative (serologic) blood group was AB, but whose red cell agglutination was atypical (“mixed field”) and caused us to study the patient’s parents’ ABO blood groups. Parental blood groups (AB and O) suggested possible nonparentage. An alternative explanation of the findings was that the child was chimeric or mosaic. Molecular cloning and genotyping of his ABO locus in leukocytes revealed two heterozygous genotypes: A102/O01 and B101/O01. Other loci, each of which possessed three distinct alleles, unambiguously showed transmission of two alleles from either the child’s mother (e.g., HLA‐A) or two alleles from the child’s father (e.g., D8S1179). Findings indicate that the child is a tetragametic chimera.

Description of two novel alleles HLA-Cw*0766 and Cw*0767 identified in a Chinese Han individual by sequence-based typing

We report two novel HLA-C alleles, Cw*0766, and Cw*0767 identified in a Chinese voluntary stem cell donor. This sample was initially found by direct PCR-SBT with an inconclusive result. To clarify this unusual SBT result, the PCR product of HLA-C gene covering from exon 1-4 was subjected to cloning and haplotype sequencing and family study of HLA inheritance consisting of six members were confirmed. Novel Cw*0766 allele differs from the closest allele Cw*07020101 by single nucleotide change at genomic DNA nt 1651 C>A (codon 206 CTG>ATG) in exon 4, which results in an amino acid change Leu206Met. Another novel allele Cw*0767 differs from the closest allele Cw*07020103 by single nucleotide change at genomic DNA nt 930 A>T (codon 161 GAG>GTG) in exon 3, which results in an amino acid change Glu161Val. Family study demonstrated that the novel Cw*0766 allele segregating on an A*2402 B*4001 Cw*0766 DRB1*1202 haplotype was inherited from the mother of the propositus, whereas the novel Cw*0767 allele on haplotype A*1101 B*0702 Cw*0767 DRB1*0101 was inherited from the father.

HLA-B*152703, a novel allele, which has arisen by silent mutation in codon 138

A novel human leukocyte antigen-B (HLA-B) allele, officially named B*152703, was found during routine high-resolution sequence-based typing in a Chinese potential hematopoietic stem cell transplantation donor. Compared with the HLA-B*152701 sequence, the B*152703 has a silent substitution at position 486(G-->C) in exon 3.

A new HLA-A*24 variant, A*2485, identified by sequence-based typing in a Chinese individual.

We report the identification of the novel allele HLA-A*2485 that was found during routine high-resolution sequence-based typing of a Chinese bone marrow donor. The A*2485 allele has 1nt change from A*240201 at nt525 where T>A, codon 151 H>Q (CAT-->CAA).

A novel HLA‐A allele detected by sequence‐based typing: HLA‐A*24:223

Compared with A*24:02:01:01, HLA-A*24:223 shows one nucleotide difference at genomic nt 149 G>A (codon 26 GGC>GAC) in exon 2.

Cloning and sequencing HLA-A and -B genomic DNA and analyzing polymorphism in regulatory regions in Chinese Han individuals: Cloning and sequencing HLA-A and -B genomic DNA and analyzing polymorphism in regulatory regions in Chinese Han individuals

In the present study, a high-resolute method for cloning and sequencing genomic full-length HLA-A and -B using 20 Chinese Han individuals was established. We detected 10 HLA-A allele sequences 4.2 kb in length and 6 HLA-B allele sequences 3.7 kb in length, and the sequences included all exons, all introns, 5promoter, and 3UTR of the two genes. All sixteen sequences have been submitted to GenBank and IMGT/HLA database. A*1153 is a novel allele, and the introns of B*151101 are firstly reported here. The 5promoter and 3UTR sequences of 5 HLA-A alleles and 2 HLA-B alleles are also firstly disclosed, and all other alleles have extended the genomic full length sequences released in IMGT/HLA database. The polymorphic structures of upper 5promoter and downstream 3UTR, which were uncovered in IMGT/HLA database, are firstly depicted in Chinese Han individuals. Twenty-six single nucleotide polymorphisms (SNPs) and one 3 bp-insertion/deletion (Indel) were located in the upper 5promoter and 14 SNPs were located in the 3UTR of HLA-A. In addition, five SNPs and one 1 bp-indel were located in the upper 5promoter and 5 SNPs were located in the 3UTR of HLA-B. Through analyzing the phylogenetic trees of 5promoter, exons and 3UTR of the two genes, we found that the evolution history of regulatory regions and exons is different between the two genes. The regulatory regions are tightly linked with exons in most of HLA-A alleles excluding A*24020101. On the contrary, recombinant events may occur frequently between regulatory regions and exons in most HLA-B alleles.

[Association of genetic polymorphisms of KIR-HLA system with chronic myeloid leukemia among ethnic Hans from southern China].

OBJECTIVEnTo explore the association of KIR-HLA gene polymorphism with chronic myeloid leukemia (CML) among ethnic Hans from southern China.nnnMETHODSnA total of 172 adult CML patients and 480 unrelated healthy controls were screened for the presence of KIR with sequence-specific primers-PCR (PCR-SSP) and sequence-based typing (SBT) of HLA-A, -B and -C loci. Polymorphisms of the KIR-HLA system were analyzed at 4 levels, and the frequencies of KIR framework genes and KIR profiles, classⅠHLA ligands, matched KIR+HLA pairs and KIR-HLA compound profile were compared between the two groups. P values were calculated using SPSS 13.0 software.nnnRESULTSnFor the CML group, the frequencies of HLA-C2 ligand, 2DL1+HLA-C2 pair and HLA-B Bw4-80I were significantly lower than those of the control group, suggesting a protective effect against CML (HLA-C2: OR=0.386, 95%CI:0.240-0.620, P<0.01; 2DL1+HLA-C2: OR=0.316, 95%CI:0.191-0.525, P<0.01; HLA-B Bw4-80I: OR=0.576, 95%CI:0.384-0.862, P<0.01). The frequencies of KIR2DL1 ligand (HLA-C2) and KIR3DL1 ligand (HLA-B Bw4-80I) in the CML group were significantly lower than that of the control group, suggesting that the HLA-C2 and HLA-B Bw4-80I expression is probably decreased in the CML patient group, which led to reduced inhibitory signal and enhanced activating signal of KIR2DL1+ and/or KIR3DL1+ NK cells. Notably, the frequency of KIR-HLA compound profiles ID2 (KIR AA1-HLA-C1/C1-Bw6/Bw6-A3/11) in CML patients significantly increased in the CML patient group compared with the control group, suggesting that the KIR-HLA compound profiles ID2 may be a risk factor for CML (OR=2.163, 95%CI 1.198-3.906, P<0.01).nnnCONCLUSIONnAbove analysis has identified certain protective and risk factors for CML from the KIR-HLA system, which may provide a clue for the pathogenesis of leukemia and development of individualized immune therapy.

Characterization of the HLA-C*07:01:01G allele group in European and African-American cohorts

The HLA-C*07:01:01G allele group consists of three nonsynonymous alleles, C*07:01:01, C*07:06 and C*07:18, plus C*07:01:02, which is synonymous to C*07:01:01. All of these alleles have identical exons 2, 3 and 4, but differ in exons 5 or 6. Therefore routine sequence-based typing (SBT) of exons 2 and 3 is unable to resolve these subtypes, resulting in ambiguous typing results in population and disease cohort studies. In the present study, we fully characterized C*07:01:01G subtypes in European and African Americans and examined their relative frequency distributions. In European Americans C*07:01:01G is predominantly represented by C*07:01:01 (94.4%), whereas C*07:01:02 (1.1%) and C*07:18 (4.5%) were detected relatively infrequently. In African Americans C*07:18 (42.4%) showed a high frequency similar to that of C*07:01:01 (44.7%) whereas C*07:06 was detected at a low frequency (4.7%). C*07:06 was found exclusively on B*44:03 carrying haplotypes in both ethnic groups, but C*07:18 showed multiple linkage relationships with HLA-B. These results demonstrate that C*07:01:01G as defined by routine SBT is a heterogeneous group of alleles, especially among individuals of African origin. If C*07:01:01G subtypes prove to bear divergent functional significance, it would be necessary to include these subtypes in routine HLA-C typing for clinical transplantation and disease association studies.